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    Home > Li Xueming of Tsinghua University and his collaborators have made great achievements in using software and traditional electron microscopy to analyze ultra-high resolution structures

    Li Xueming of Tsinghua University and his collaborators have made great achievements in using software and traditional electron microscopy to analyze ultra-high resolution structures

    • Last Update: 2019-08-29
    • Source: Internet
    • Author: User
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    Recently, Li Xueming's research group and collaborators from the school of life, Tsinghua University published a paper entitled "programming conventional electron microscopes for solving ultra-resolution structures of small and large molecules" in Analytical Chemistry Research paper of macro molecules (DOI: 10.1021/acs.anlchem.9b01162)

    This research has released a new micro ed data collection software etased, which can analyze the polypeptide and small molecule structure to about 0.6 μ m under the traditional 200 kV and 120 kV transmission electron microscope equipped with a single frame camera, and can clearly see the density of almost all hydrogen atoms

    This study eliminates the dependence of micro ed on high-end camera with continuous photo taking mode, proves the availability of 120 kV low-end electron microscope in micro ed, greatly reduces the equipment cost of micro ed, improves the general availability of this method, and will contribute to the wider application of micro ed technology

      In recent years, micro ed technology has been widely used to analyze the crystal structure of proteins and small molecules

    However, at present, all research groups need to rely on high-end cameras with continuous photographing mode for data collection of micro ed, so that the camera can take continuous pictures during the continuous rotation of the sample table, so as to avoid the lack of information generated by the reconstructed three-dimensional lattice in the inverted space, leading to structural analysis Error

    However, except for special needs, high-end cameras with continuous photographing mode are generally not installed on the relatively low-end 200 kV or 120 kV electron microscope, which leads to the need to upgrade the hardware to carry out micro ed research, and the additional cost hinders the wide application of micro ed technology

    In addition, according to the optical principle of the electron microscope, the electron diffraction is not restricted by the resolution of contrast transfer function (CTF) like the image, so theoretically the low-end 120 kV electron microscope can also be used for the electron diffraction analysis at the sub angstrom resolution level

    However, most of the current researches on micro ED are made by using 200 kV TEM equipped with field emission electron gun, and the availability of 120 kV TEM also needs to be explored

    The research team and collaborators of Li Xueming's research group have developed etased software, which synchronizes the behavior of the camera and the sample table through the software, so that the sample table only rotates within the actual effective exposure time of the camera, and remains stationary for the rest of the time to wait for the camera to be ready, so that the data collected by the single frame camera can also completely cover the switching space without information loss

    The research team used Kikuchi line, a diffraction pattern with invariable translation, as a ruler to test the stability of the sample table rotation under the experimental conditions

    The test shows that the sample table rotation mode adopted by the software has high stability, and the acceleration and deceleration process is very short, and its impact can be ignored under relatively long exposure time

    With the help of etased software, the research team used 200 kV Fei F20 electron microscope and 120 kV Fei T12 electron microscope to analyze the microcrystalline sample of a polypeptide to 0.65 Å and 0.60 Å, respectively

    For the first time, all hydrogen atoms were clearly located in the macromolecular structure, and the complex hydrogen bond network in the crystal structure could be directly observed, which is of great significance for the study of ultra-high resolution structure of biological macromolecules

    In addition, the team also analyzed small molecular compounds acetaminophen to 0.65 Å (F20) and 0.83 Å (T12), and biotin to 0.75 Å (F20) and 0.90 Å (T12)

    The density of almost all hydrogen atoms can also be directly seen in the acetaminophen structure with a resolution of 0.65 μ M

    This research reduces the requirements of micro ed data collection on hardware, so that researchers can use the existing hardware around them to collect high-quality micro ed data in low cost and analyze the ultra-high resolution structure, which can promote the wider and deeper application of micro ed technology in the field of life science and pharmaceutical chemistry

    Li Xueming, researcher of School of life, Tsinghua University, and Liu Cong, researcher of biology and chemistry cross research center, Shanghai Institute of organic chemistry, Chinese Academy of sciences are the co authors of this paper

    Zhou Heng, 2015 Ph.D

    student, School of life, Tsinghua University, Luo Fengbo, Institute of Organic Chemistry, Chinese Academy of Sciences, and Luo Zhipu, School of life, Tsinghua University Doctor (now professor of Molecular Enzymology Institute, Suzhou University) is the co-author of this paper

    The frozen electron microscope platform of Tsinghua University, national Protein Science Center (Beijing) provided support for sample preparation and data collection

    The National Natural Science Foundation of China, Beijing advanced innovation center of structural biology and Tsinghua Peking University Life Science Joint Center provided financial support for this study.
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